Bacterial pathogens are a common cause of serious disease in farm animals and household pets. The majority of such diseases are caused by various bacterial species, including but not limited to Pasteurella multocida. P. haemolytica, Haemophilus somnus, H. pleuropneumoniae, Bordetella bronchiseptica, Moraxella bovis, and Erysipelothrix rhusiopathiae. Standard vaccines for these disease fall into a class of vaccines called bacterins, which are inactivated or attenuated whole organism preparations. The efficacy of bacterins, however, has been questioned. See, e.g., Hall, R. F., et al, 1977, Vet. Med. Small Animal Clin. 72: 1368-1370. Moreover, the immune response to bacterins is generally short-lived and is not directed to specific cellular constituents associated with the virulence of the microorganism.
One possible alternative to bacterins is production of a vaccine based upon specific immunogenic components isolated from the pathogenic organism. Although the isolation of specific bacterial components is well known in the art, economical and practical veterinary vaccines based on such components have not been thought possible. See, for example, Rosendal et al. (1986, Vet. Microbiol. 12: 229-240) who describe a process of bacterial cell extraction from Haemophilus pleuropneumonia using sodium chloride and Cetavalon (hexadecyltrimethyl ammonium bromide) but conclude that such extractions are not sufficiently efficacious as a vaccine to warrant field use. Other work on acellular vaccines include Adlam et al. (1984, J. Gen. Microbiol. 130: 2415-2426, describing a process for obtaining purified capsular polysaccharide by a series of alcohol and acetone extractions) and McKinney et al. (1985, Vet. Microbiol. 10: 465-480, describing a process of saline extraction of the bacteria followed by separation of heterogenous protein components by preparative and analytical isoelectrofocusing).
Vedros et al. (1982, 13th Ann. Conf. Aquatic Animal Med.) reported preparation of an acellular vaccine for pasteurellosis (Pasteurella multocida) in marine mammals. That process comprised shearing away surface components of the cell with glass beads; precipitation of the components using ammonium sulfate; removal of proteins using enzymes and alkaline hydrolysis; and separation of the polysaccharide components through alcohol precipitation, molecular sieving and ion exchange chromatography. A modification of this process was used to produce an acellular vaccine for melioidosis (Pseudomonas pseudomallei) in marine mammals (Vedros et al., 1988, Dis. Aquatic Org. 5: 157-161). Further, U.S. patent application No. 07/081,942, filed Aug. 5, 1987, now U.S. Pat. No. 4,877,613, describes a process for preparing acellular vaccines against gram negative non-enteric pathogenic bacilli, in which surface and cellular polysaccharides are mechanically sheared away, followed by precipitation with Cetavalon, removal of nucleic acids with alcohol, and then further precipitated with alcohol yielding "Formulation I." The supernatant of the Cetavalon precipitation was further treated with alcohol to yield "Formulation II." The preferred vaccine was derived from a mixture of Formulations I and II.
In the area of human medicine, acellular vaccine preparations are known, but are derived through complicated and expensive procedures. Such vaccines have been disclosed in U.S. Pat. Nos. 3,636,192 to Gotschlich; No. 4,753,796 to Moreno; and No. 4,755,381 to Cryz. Further examples can be found in Gotschlich, 1975, Monograph. Allergy 9: 245-258. These references disclose methods for obtaining specific cellular components which previous research has shown to be immunogenic, but require the use of quaternary ammonium salts in combination with alkanols to precipitate the desired polysaccharides. In cases where endotoxins are present, further extensive and complicated purification is necessary.
Although the art reveals certain known methods for preparing acellular vaccines, such methods are inadequate or unsatisfactory in one or more ways. Many of these techniques are complicated and expensive requiring many man hours to produce efficacious vaccines.
Another difficulty with known methods lies in the culture media of the bacterium of interest. Standard culture media include a wide array of components designed to foster abundant bacterial growth. Not only do such formulations tend to be expensive, and thereby add to vaccine cost, but they also include high molecular weight compounds such as animal sera that are very difficult to remove during vaccine purification. However, it is widely believed that such formulations are indispensable in order to ensure growth sufficient for vaccine production.
Known methods also require the use of large quantities of alkanols and other volatile compounds that are dangerous and costly to both store and use.
Finally, known methods of preparing acellular vaccines use live as opposed to killed microorgansims, necessitating laborious sterilization and isolation techniques as well as constant maintenance of cultures in order to ensure an adequate supply of fresh individuals.